Rapid Quantitative Detection And Genotyping Of Staphylococcus Aureus In Frozen Flour And Rice Products

In this study,retailed frozen flour and rice products were collected from supermarkets in Shanghai.Staphylococcus aureus was isolated and identified according to the national standard method(GB4789.10-2010),and then the isolates were further confirmed as S.aureus by PCR amplification of the nuc1 gene.Four DNA extraction methods(CTAB method,spin column kit method,amino-modified silica-coated magnetic nanoparticles method and carboxyl beads kit method)were compared to find an optimum DNA extraction method from artificially contaminated frozen flour and rice products without pre-enrichment.And a new rapid quantitative detection method for the S.aureus contamination in food samples was built by combining nano-magnetic separation with TaqMan Real-time PCR.All the S.aureus isolates acquired in this study were screened for their enterotoxin gene diversity by PCR amplification of 18enterotoxin genes,and combining with the Multilocus Sequence Typing(MLST)results,the enterotoxin distribution in different genotypes were analyzed.(1)In total,288 frozen flour and rice products samples(meat-stuffing,vegetable-stuffing and non-stuffing)were collected to detect the contamination levels of S.aureus according to the national standard method(GB4789.10-2010).And the PCR amplification of nuc1 gene was performed for further confirmation of the presence of S.aureus.It turned out that S.aureus existed in 88 samples out of 288,so that the total positive rate was30.56%.There was remarkable difference between 3 kinds of frozen flour and rice products,and the meat-stuffing samples had the highest rate of41.44%.After confirmation,124 isolates were acquired from the 88 positive samples.(2)Four DNA extraction methods(CTAB method,spin column kit method,amino-modified silica-coated magnetic nanoparticles method and carboxyl beads kit method)were compared for the extracted DNA quantity and purity from artificially contaminated frozen flour and rice products by S.aureus without pre-enrichment.It showed that amino-modified silica-coated magnetic nanoparticles method was significantly more efficient than the others for higher quantity and purity of DNA,which can eliminate proteins and lipids effectively and complete the whole DNA extraction workflow in 1.5 h.(3)A new rapid quantitative detection method by combining amino-modified silica-coated magnetic nanoparticles method with Taq Man Real-time PCR was developed so that the quantitative detection results within 4h.The new quantitative detection method was compared with the reference BP plating method in 32 S.aureus positive samples,and it demonstrated that there was no remarkable difference(p>0.05)between the two methods.(4)Based on the rapid quantitative method established in this study and national standard method,the contamination level of S.aureus in quick frozen flour and rice products was analysed.It demonstrated that the meat-stuffing samples had the highest contamination level which ranged from2.00×10~3 to 1.21×10~5 CFU/g,while in the vegetable-stuffing and non-stuffing samples,the amount of S.aureus was 7.88×10~2~7.89×10~3 CFU/g and 4.68×10~2~5.60×10~3 CFU/g respectively.Six out of 88 positive samples exceeded the allowable limit in the new national standard(10~4 CFU/g)(5)The distribution of 18 enterotoxin genes(sea~see,seg~ser and seu)in the 124 S.aureus isolates was screened.Among the 124 isolates,111were positive for enterotoxin genes with the total carrying rate of 89.52%.Among these positive isolates,5 contained 10 enterotoxin genes with the biggest carrying number.No enterotoxin genes were found in 13 isolates.And the sep gene was the most detected(44.35%,55/124),followed by sei(43.55%,54/124)and seg(42.74%,53/124).The detection rates of these 3enterotoxin genes were exceeded 40%.(6)In order to identify the genetic correlation among these S.aureus isolates,MLST typing was carried out.It illustrated that the isolates were scattered in 36 STs including 31 already-known ST types and 5 new suspicious ST types(new-1~new-5).ST-7 represented the largest group of27 isolates,followed by ST-5 and ST-25 with 7 isolates,respectively.ST-9,ST-15,ST-2871,and ST-188 were also very common with more than 5isolates.Further more,these 36 ST types were attributed to 19 CCs by eBURST analysis.CC7 was the most prevalent clonal complex accounted for 29.84%(37/124).CC7(ST7,ST306 and ST943)and CC239(ST630,ST239 and ST3159)were more diverse for containing more ST types than the others.Combining the results of enterotoxin gene screening and MLST typing,it was found that ST-7 had the most isolates and the enterotoxin genes of all the ST types.The average numbers carrying enterotoxin genes in CC30,CC9,CC72,CC5 and CC25 were higher than the others.And the five traditional enterotoxin genes(sea~see)were almost distributed in 4 CCs(CC1,CC5,CC25 and CC7).In this study,Staphylococcus aureus was identified and isolated from frozen flour and rice products and a new rapid quantitative detection was built up followed with enterotoxin screening and MLST typing.It can lay the foundation for rapid quantitative detection of Staphylococcus aureus in food,as well as it can provide theoretical direction and data support for food safety monitoring and risk assessment.